The present invention relates to the cloning and sequencing of the biosynthetic gene cluster that encodes a Type I polyketide synthase (PKS) and a non-ribosomal peptide synthase responsible for the production of meridamycin. The present invention also relates to methods for genetically manipulating the meridamycin biosynthetic pathway to produce derivatives of meridamycin.
Polyketides represent a large group of natural products that are derived from successive condensations of simple carboxylates, such as acetate, propionate or butyrate. Naturally occurring polyketides possess a broad range of biological activities, including antibiotics such as tetracyclines and erythromycin, anticancer agents such as daunomycin and bryostatin, immunosuppressants such as FK506 and rapamycin, and veterinary products such as monensin and avermectin. Polyketides are produced in most groups of organisms and are especially abundant in a class of mycelial bacteria, the actinomycetes, which produce various types of polyketides.
The enzymes responsible for the biosynthesis of polyketides are called polyketide synthases (PKSs). Two general classes of PKSs exist. One class, known as Type I PKSs, is represented by the PKSs for the synthesis of macrolide polyketides such as erythromycin and rapamycin. This type of PKSs has a modular enzymatic structure, in which a module is defined as a set of enzymatic domains that are necessary to catalyze the recognition and incorporation of a specific 2-carbon extending unit (usually a malonyl-CoA, a methyl malonyl-CoA or a propionyl-CoA) into the growing polyketide chain. A minimal type I PKS module contains three enzymatic domains: (1) a ketosynthase domain (KS) which is responsible for catalyzing the Claisen condensation reaction between a starter unit or a growing polyketide chain and an extender unit; (2) an acyltransferase domain (AT) which selectively binds a specific extender unit from the intracellular pools of the various CoA carboxylates and then transfers it to the acyl carrier center; (3) an acyl carrier protein domain (ACP) which contains a serine residue that has been post-translationally modified with a 4-phosphopantethein group and serves as the acceptor for the extender unit or a growing polyketide chain. In addition to the KS, AT, and ACP domains, a type I PKS module can also have one, two or three of the following domains: a ketoreductase domain (KR) which reduces the β-ketone to the hydroxyl function, a dehydratase domain (DH) which eliminates water from the α, β carbon centers to generate a double bond between them, and a enoylreductase domain (ER) which further reduces the double bond generated by DH domain to yield the β-methylene group.
A co-linear relationship exists between the primary organization of the Type I PKS and the structure of the polyketide backbone. For examples, the number of modules in the PKS determines the number of the two-carbon units in the carbon backbone of the final polyketide product, the presence of a specific AT domain determines which extender (malonate, methylmalonate or ethylmalonate, etc.) is incorporated into the growing polyketide chain, and the presence of the reduction domains (KR, DH and ER) in a module determines the extent of reduction of the β-carbon formed at the give condensation.
The second class of PKSs, called Type II PKSs, is responsible for the synthesis of aromatic polyketides such as daunorubicin and tetracenomycin. Type II PKSs have a single set of enzymatic activities (KS, AT, ACP, KR etc.) that reside in individual proteins and are used iteratively to generate polyketides with polycyclic ring structure. There is no clear correlation between the type II PKS enzymatic organization and the final polyketide structure.
The genes encoding PKSs and the necessary tailoring enzymes to make a polyketide compound have been shown in all cases to be clustered together on the chromosome of the producing microorganism, and thus are collectively called “PKS biosynthetic gene cluster”. Tremendous research work has been done in academic and industrial fields aimed at generating novel polyketide compounds with potential therapeutic applications through genetic manipulation of PKS biosynthetic gene clusters. There is a continuing need in the art to determine the genes encoding novel PKS complexes.